Plant transgenic work is beset with low and inconsistent levels of transgene expression. Episomal vectors are expected to overcome these problems. In microbes, episomal (plasmid) vectors are possible because these vectors can be maintained by selection. Although plant viruses have been used as episomal expression vectors, their use has been restricted to transient expression because of lack of selection and/or their cellular toxicity (U.S. Pat. No. 4,855,237, WO 9534668).
Plant viruses
Viruses are infectious agents with relatively simple organization and unique modes of replication. A given plant virus may contain either RNA or DNA, which may be either single- or double-stranded.
Rice dwarf virus (RDV) and wound tumor virus (WTV) are examples of double-stranded RNA plant viruses. Single-stranded RNA plant viruses include tobacco mosaic virus (TMV), turnip yellow mosaic virus (TYMV), rice necrosis virus (RNV) and brome mosaic virus (BMV). The RNA in single-stranded RNA viruses may be either a plus (+) or a minus (-) strand. Although many plant viruses have RNA genomes, organization of genetic information differs between groups. The genome of most monopartite plant RNA viruses is a single-stranded molecule of (+)-sense. There are at least 11 major groups of viruses with this type of genome. An example of this type of virus is TMV. At least six major groups of plant RNA viruses have a bipartite genome. In these, the genome usually consists of two distinct (+)-sense single-stranded RNA molecules encapsidated in separate particles. Both RNAs are required for infectivity. Cowpea mosaic virus (CPMW) is one example of a bipartite plant virus. A third major group, containing at least six major types of plant viruses, is tripartite, with three (+)-sense single-stranded RNA molecules. Each strand is separately encapsidated, and all three are required for infectivity. An example of a tripartite plant virus is alfalfa mosaic virus (AMV). Many plant viruses also have smaller subgenomic mRNAs that are synthesized to amplify a specific gene product. Plant viruses with double-stranded DNA genome include Cauliflower Mosaic virus (CaMV).
Geminiviruses
Plant viruses with single-stranded DNA genomes are represented by geminiviruses and include African Cassava Mosaic Virus (ACMV), Tomato Golden Mosaic Virus (TGMV), and Maize Streak Virus (MSV). Geminiviruses are subdivided on the basis of whether they infect monocots or dicots and whether their insect vector is a leafhopper or a whitefly. Subgroup I geminiviruses are leafhopper-transmitted that infect monocotyledonous plants (e.g., Wheat Dwarf Virus), Subgroup II geminiviruses are leafhopper-transmitted that infect dicotyledonous plants (e.g., Beet Curly Top Virus), and Subgroup III geminiviruses are whitefly-transmitted that infect dicotyledonous plants (e.g., Tomato Golden Mosaic Virus, TGMV, and African Cassava Mosaic Virus, ACMV). Subgroup I and II geminiviruses have a single (monopartite) genome. Subgroup III geminiviruses have a bipartite genome. For example, TGMV and ACMV consist of two circular single-stranded DNA genomes, A and B, of ca. 2.8 kB each in size. DNA of genome A and DNA of genome B of a given Subgroup III virus have little sequence similarity, except for an almost identical common region of about 200 bp. While both DNA of genome A and DNA of genome B are required for infection, only DNA of genome A is necessary and sufficient for replication and DNA of genome B encodes functions required for movement of the virus through the infected plant.
In both TGMV and ACMV, DNA A contains four open reading frames (ORFs) that are expressed in a bidirectional manner and arranged similarly. The ORFs are named according to their orientation relative to the common region, i.e., complementary (C) versus viral (V) in ACMV and leftward (L) or rightward (R) in TGMV. Thus, ORFs AL1, AL2, AL3, and AR1 of TGMV are homologous to AC1, AC2, AC3, and AV1, respectively, of ACMV. Three major transcripts have been identified in ACMV DNA A and these map to the AV1 and AC1 ORFs, separately and the AC2/AC3 ORFs together. There is experimental evidence for the function of these ORFs. Thus, in ACMV AC1 encodes a replication protein that is essential and sufficient for replication, AC2 is required for transactivation of the coat protein gene, AC3 encodes a protein that is not essential for replication but enhances viral DNA accumulation, and AV1 is the coat protein gene. Except for the essential viral replication protein (encoded by AC1 and AL1 in ACMV and TGMV, respectively), geminivirus replication relies on host replication and transcription machinery. Although geminiviruses are single-stranded plant DNA viruses, they replicate via double-stranded DNA intermediate by `rolling circle replication`.
Viruses as Expression Vectors
Construction of plant viruses to introduce and express non-viral foreign genes in plants has been demonstrated (U.S. Pat. No. 4,855,237, WO 9534668). When the virus is a DNA virus, the constructions can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all of the constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral RNA. Alternatively, the cDNA can be cloned behind a heterologous plant promoter, introduced into a plant cell, and used to transcribe the viral RNA that can replicate autonomously [Sablowski et al. (1995) Proc. Natl. Acad. Sci. USA vol 92, pp 6901-6905].
Geminiviruses have many advantages as potential plant expression vectors. These include 1) replication to high copy numbers nonsymptomatically, 2) small, well-characterized genomes, 3) assembly into nucleosomes, and 4) nuclear transcription. The DNA A component of these viruses is capable of autonomous replication in plant cells in the absence of DNA B. Vectors in which the coat protein ORF has been replaced by a heterologous coding sequence have been developed and the heterologous coding sequence expressed from the coat protein promoter [Hayes et al. (1989) Nucleic Acids Res. vol. 17, pp. 2391-403; Hayes et al. (1988) Nature (London) vol. 334, pp. 179-82].
Greater than fill length copies of wild type TGMV A and B genomes were transformed into petunia [Rogers et al. (1986) Cell (Cambridge, Mass.) vol. 45, pp. 593-600]. Replication was reported in the primary transformants and in some of the selfed progeny consistent with its mendelian inheritance, indicating that the chromosomally-integrated master copy, not the replicon, is inherited. This suggests that gametophytic and/or developing seed tissues lack the ability to support replication. The report did not demonstrate whether the virus replicated in non-germinating seed tissue. Prior art shows that geminiviruses are not seed-transmitted in nature [Goodman, (1981) J. Gen. Virol. vol. 54, pp. 9-21]. Thus, there was no evidence that they can replicate in gametophytic tissue or developing seed.
Tomato Golden Mosaic Virus (TGMV) DNA A was modified by replacing its coat protein coding sequence with that of NPT II or GUS reporter genes or with that of 35S:NPT II gene and a greater than full length copy of the modified viruses were transformed into tobacco [Hayes et al. (1989) Nucleic Acids Res. vol. 17, pp. 2391-403; Hayes et al. (1988) Nature (London) vol. 334, pp. 179-82]. Leaves of transgenic plants showed that the high levels of the reporter enzymes was gene copy number-dependent. However, replication of the vector and reporter gene expression were not reported in seed and the genetic stability of the vector in transgenic plants in subsequent generations was not reported. Use of the African Cassava Mosaic Virus (ACMV) in similar fashion has not been reported and it is not known that ACMV DNA or the replication protein(s) can be stably maintained in progeny plants and whether it can replicate in seed tissues.
In one report, a chimeric gene in which the constitutive plant promoter, 35S, was fused to the TGMV sequence containing ORFs AL1, AL2, and AL3 were transformed into Nicotiana benthamiana. Different transgenic lines showed significant non-uniformity in the levels of 35S:AL1-3 gene expression as well as their ability to complement viral replication [Hanley-Bowdoin et al. (1989) Plant Cell vol. 1, pp. 1057-67]. In another report, chimeric genes in which the constitutive plant promoter, 35S, was fused to the coding sequence of TGMV replication protein AL1 were transformed into tobacco. The expression of TGMV replication protein in the primary transformants supported the replication of a mutant genome A lacking the replication protein. [Hanley-Bowdoin et al. (1990) Proc. Natl. Acad. Sci. U.S.A. vol. 87, pp. 1446-50]. However, in both reports neither the genetic stability of the chimeric replication protein gene through subsequent generations nor its ability to support viral replication in seed tissue was reported. In another report, chimeric genes in which the constitutive plant promoter, 35S, was fused separately to the coding sequences of TGMV replication proteins AL1, AL2, and AL3 were transformed into tobacco [Hayes et al. (1989) Nucleic Acids Res. vol. 17, pp. 10213-22]. The TGMV replication protein was shown to have been expressed in progeny but the genetic stability of the chimeric replication protein gene through subsequent generations was not reported. Furthermore, it was not reported whether the transgenic plants will support replication in seed tissue.
In another disclosure, Rogers et al. (EP 221044) demonstrated the expression of foreign proteins in plant tissue using a modified "A" genome of the TGMV gemini virus. The foreign gene was inserted in place of the gene encoding the viral coat protein and the resulting plasmid transformed into plant tissue. Rogers et al. did not report tissue specific expression of the foreign protein and are silent as to the genetic stability of the transforming plasmid.
All of the reported viral vectors have a major disadvantage. They were either not shown to be stably maintained in transgenic plants and/or not practically useful. Thus, despite intense efforts to develop plant viral vectors and viruses, no commercially useful plant virus-based recombinant vectors have been developed that are heritable and capable of episomal replication and expression in desired tissue(s) of the transgenic host plant without the need for infection every generation. In fact, replication of plant viruses is expected to be detrimental to the growth and development of plant cells. For example, when greater than full length copy of TGMV genome A is introduced into plant cell one-tenth as many transgenic plants are obtained than when genome B is used or when control transformations are done [Rogers et al. (1986) Cell (Cambridge, Mass.) vol. 45, pp. 593-600]. The authors suggest that this may be due to expression of a gene in TGMV A DNA. Furthermore, crude extract of plants expressing tandem copies of both TGMV A and TGMV B genomes are unable to infect Nicotiana benthamiana plants. This is consistent with having a low virus titer. Thus, transgenic plants that do regenerate could be selected for low level expression of a toxic viral gene product and low level of viral replication. This is also consistent with the authors' finding that relatively few cells initiate release of the virus, a conclusion based on their observation that most of the tissues remain viable and nonsymptomatic. Similarly, poor replication in transgenic plants containing 35S:replication protein in other reports suggest that plants are either selected for poor expression of the replication protein, presumably because of its toxicity, or that the tissue-specific expression profiles of the replication gene is different from that of viral replication.
Recently, it was reported that 6 of 11 transgenic tobacco plants stably transformed with a monopartite geminivirus (Tobacco Yellow Dwarf Virus) with a functional replication gene showed episomal replication [Needham et al. (1998) Plant Cell Rep. vol. 17, pp. 631-639].
Silencing endogenous genes and transgene is an important technology [see Senior et al. (1998) Biotechnol. Genet. Eng. Rev. vol. 15, pp. 79-119; Thomas et al. (1998) Plant Growth Regul. vol. 25, p. 205]. Silencing of endogenous genes or transgenes by viral infection or by stably incorporated virus in transgenic plants has been reported for RNA virus [Baulcombe et al. PCT Int. Appl. (1998), Ruiz et al. (1998) Plant Cell vol. 10, pp. 937-946], Geminiviruses [Kjemtrup et al. (1998) Plant J. vol. 14, pp. 91-100, Atkinson et al. Plant J. vol. 15, pp. 593-604], and Cauliflower Mosaic Virus [Al-Kaff et al. Science (Washington, D.C.) 279:2113-2115 (1998)]. However, regulated virus induced silening in transgenic plants, which is expected to provide regulated gene silencing, has not been reported.
To date, no plant virus-based recombinant vectors are known that are heritable and capable of episomal replication and expression of foreign proteins in target tissue(s) of a transgenic host plant without the need for infection in every generation.